1. Field of the Invention
The invention relates to bifunctional glycoproteins having targeting protein and enzyme properties. More particularly, the invention relates to such proteins whose complements of carbohydrate residues have been modified in a manner that enhances the clearance of such proteins from the circulation and increases the relative binding of the proteins at the tumor site. The enzymatic portion is capable of converting a prodrug into a cytotoxic drug that attacks tumor cells.
This invention also relates to the treatment of tumors with such proteins, and the production of such proteins, including recombinant production and production by transgenic animals.
2. Description of the Background Art
In efforts to control tumors, attempts have been made in the last twenty years to achieve selective therapeutic effects based on the specificity of antibodies. However, important therapeutic successes still have not been achieved in the case of solid tumors. Although highly specific tumor-selective monoclonal antibodies are available for targeting purposes, the lack of success in immunotherapy is primarily due to the small quantities of monoclonal antibody molecules that can be localized to solid tumors. One reason for this low degree of localization, which is generally insufficient for therapeutic purposes, is the presence of diffusion barriers in the tumor (Jain, R. K., Cancer Res. 47: 3039 (1987)). Prior attempts to compensate by increasing the dosage of the drug have encountered problems of widespread non-specific binding in non-tumor structures, and generalized toxic side effects.
Prior art compounds have sought to utilize (i) the specificity of a monoclonal antibody or tumor-binding protein partner and (ii) the catalytic amplification potential of an enzyme. Such antibody-enzyme conjugates can be administered to a patient and given time to bind to the tumor. Thereafter, a non-toxic prodrug, which can be cleaved by the enzyme portion of the conjugate to yield a cytotoxic drug, is administered to the patient. In theory, the enzyme portion of the molecules bound to the tumor converts the prodrug in the vicinity of the tumor into a drug which is cytotoxic to the tumor. In reality, however, such compounds suffer several drawbacks.
First, such antibody-enzyme conjugates are highly immunogenic in humans, since they represent chemical conjugates composed, as a rule, of mouse antibodies and xenogeneic enzymes. Repeated use of the same antibody-enzyme conjugate on the same patient is therefore not possible clinically (Bagshawe et al., Disease Markers, 9: 233 (1991)).
Second, the conjugates are only relatively slowly removed from the plasma, so that selective and effective prodrug activation is only possible if the elimination of the unwanted non-bound enzyme activity from the plasma is significantly enhanced.
The above-mentioned problem of the immunogenicity of xenogeneic antibody-enzyme conjugates is largely solved by using a recombinant fusion protein that is composed of purely human components. Details for the production of such fusion proteins are described in European Patent Application EP-A-0 501 215, which is incorporated by reference to the extent that it discloses such fusion proteins. In that publication, proteins are described, for example, of the general formula hutuMab-L-β-gluc, with hutuMab being a humanized, or human, tumor-specific monoclonal antibody, or a part thereof which still binds to the tumor, L representing a linker moiety, and β-gluc denoting human β-glucuronidase.
However, in carrying out pharmacological tests on such a fusion protein, it was unexpectedly found that, even at very short periods Cf time (1-3 minutes) after i.v. injection of the fusion protein into human tumor-carrying nude mice, significant quantities of the protein were bound to tumor cells in regions which are close to the blood vessels (easily accessible sites=EAS). Further, at these early times, large quantities of the fusion protein were still present in the plasma, so that selective and effective activation of a suitable prodrug in the tumor was not possible at this early time point after injection.
One proposed solution to the above problem is described in the International Patent Application WO 89/10140, which discloses a three component system for treatment of malignant diseases. The first component localizes at the tumor and has enzymatic activity, e.g., an antibody-enzyme conjugate. The second component is able to bind to the first component and inactivate the catalytic site and/or accelerate the clearance of the first component from the plasma. The third component is a prodrug which can be converted by the enzymatic activity of the first component to form the cytotoxic substance used to treat the tumor.
The three components of 89/10140 are designed to be administered sequentially, not simultaneously. That is, the first component is administered and given sufficient time to localize-at the tumor site. The second component is then administered after the first component has localized. Later, the third component is given.
The process of WO 89/10140 has drawbacks, however. First, it has the disadvantage of being a more complex process than the two step processes of the prior art. Moreover, by introducing an additional substance to the human or animal body, particularly the second compound, the risk of side effects and/or adverse reactions, such as the development of an unwanted immune reaction, is increased.
Accordingly, there remains a need for improved compounds and methods for selectively targeting tumors with cytotoxic drugs.